Project Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major public health problem worldwide and the most common presenting illness among people living with HIV accounting for one in four HIV/AIDS-associated deaths. The continuing rise of multidrug-resistant strains of Mtb makes the development of new effective anti-TB drugs a high priority. In this context, re-examining the molecular mechanism of action of established antitubercular drugs that target the same well-validated biosynthetic pathway as front-line anti- TB agents but at a distinct catalytic step, thereby bypassing widespread resistance mechanisms, could have a major impact on the treatment of drug-resistant TB. Two such drugs previously used in the clinical treatment of TB, Isoxyl (ISO) and Thiacetazone (TAC), are the object of our investigations. ISO and TAC are thiocarbamide-containing prodrugs that require activation of their thiocarbonyl moiety by the Mtb flavin-dependent monooxygenase EthA for bactericidal activity. Following activation, ISO and TAC inhibit the biosynthesis of mycolic acids thereby abolishing the formation of the outer membrane of Mtb and leading to cell death. We recently identified the dehydration step of the type II fatty acid synthase (FAS-II) elongation cycle as the point at which both ISO and TAC inhibit the biosynthesis of mycolic acids. Specifically, we found that ISO and TAC block the FAS-II elongation cycle by covalently modifying the essential FAS-II dehydratase, HadAB. Despite this important breakthrough, it is still unclear whether ISO and TAC also inhibit the (non- essential) FAS-II dehydratase HadBC, and the mechanism(s) through which missense mutations in HadC increase 16 to 32-fold the resistance of Mtb to both drugs remain(s) unexplained. This application is to complete our understanding of the mechanisms of susceptibility and resistance of Mtb to ISO and TAC with the long-term goal of developing novel inhibitors of the dehydration step of FAS-II with reduced resistance frequencies and much improved potency, specificity and pharmacological features than ISO and TAC. Specifically, we propose under Aim 1 to determine whether ISO and TAC inhibit HadBC in vitro and in whole Mtb cells and, in Aim 2, to define the molecular mechanisms through which HadC missense mutations confer resistance to both drugs.